Enhancing bone regeneration: Unleashing the potential of magnetic nanoparticles in a microtissue model

Abstract Bone tissue engineering addresses the limitations of autologous resources and the risk of allograft disease transmission in bone diseases. In this regard, engineered three‐dimensional (3D) models emerge as biomimetic alternatives to natural tissues, replicating intracellular communication. Moreover, the unique properties of super‐paramagnetic iron oxide nanoparticles (SPIONs) were shown to promote bone regeneration via enhanced osteogenesis and angiogenesis in bone models. This study aimed to investigate the effects of SPION on both osteogenesis and angiogenesis and characterized a co‐culture of Human umbilical vein endothelial cells (HUVEC) and MG‐63 cells as a model of bone microtissue. HUVECs: MG‐63s with a ratio of 4:1 demonstrated the best results among other cell ratios, and 50 μg/mL of SPION was the optimum concentration for maximum survival, cell migration and mineralization. In addition, the data from gene expression illustrated that the expression of osteogenesis‐related genes, including osteopontin, osteocalcin, alkaline phosphatase, and collagen‐I, as well as the expression of the angiogenesis‐related marker, CD‐31, and the tube formation, is significantly elevated when the 50 μg/mL concentration of SPION is applied to the microtissue samples. SPION application in a designed 3D bone microtissue model involving a co‐culture of osteoblast and endothelial cells resulted in increased expression of specific markers related to angiogenesis and osteogenesis. This includes the design of a novel biomimetic model to boost blood compatibility and biocompatibility of primary materials while promoting osteogenic activity in microtissue bone models. Moreover, this can improve interaction with surrounding tissues and broaden the knowledge to promote superior‐performance implants, preventing device failure.


| INTRODUC TI ON
Significant bone defects caused by trauma, tumours and infection are generally considered critical clinical challenges as they cause a delayed union or nonunion in bones.This can compromise musculoskeletal function, leaving surgical techniques such as autografting the only available option. 1,2However, autografting is restricted due to substantial drawbacks such as limited graft supply, donor complications and disease transmission.In this regard, bone tissue engineering has been demonstrated to be promising in providing autograft alternatives such as in-vitro bone scaffolds as well as tissueengineered microtissues mimicking the body's natural systems. 1 Significantly, three-dimensional (3D) cell-culture systems are eagerly anticipated to remodel the physiological environment, improving the diffusion and adhesion of essential elements, including proteins, growth factors and enzymes.This, in turn, ensures cell viability and promises the model's function. 3spite significant advancements in this area over the past 20 years, bone tissue engineering techniques have yet to be used in clinical settings.This is primarily because of a lack of blood flow to the implanted location as well as the engineered tissue construct. 4ood vessel development (angiogenesis) is crucial for osteogenesis, the process of bone generation, as the osteoblasts can't survive, and the process of bone repair will be stopped without an appropriate blood supply. 1,5Hence, simulating the physiological bone tissue environment to create optimal conditions for developing new microvessels within bone structures would greatly assist the issue.In this context, endothelial cells and osteoblasts interacting in a co-culture setting to model an in-vitro bone microtissue may bring promising outcomes. 6e complex anatomy of bone tissue has made regenerating this organ more complicated.For example, bone tissue has special mechanical characteristics that make it challenging for tissue engineering. 7,8Recent studies have shown how the unique properties of super-paramagnetic iron oxide nanoparticles (SPIONs) would be applicable to support bone tissue regeneration.Superparamagnetic iron oxide agents have previously been used as a magnetic resonance contrast in clinical trials.Ferumoxytol, a super-paramagnetic iron oxide family member, has been approved for treating adult patients with chronic renal disease who are irondeficient. 9Its property in combating iron deficiency is due to its capability to contribute to iron homeostasis by being digested by ionization into iron ions. 10,11 a bone tissue regenerating investigation, Fe 3 O 4 nanoparticles (as a type of SPIONs) have been illustrated to promote the nanocomposite scaffold strength, calcium deposition and alkaline phosphatase (ALP) activity.3][14][15] A static magnetic field (SMF) improved cartilage extracellular matrix and chondrogenesis, alleviating osteoarthritis in mice models.Moreover, it stimulated endogenous stem cell migration by triggering the Piezo1-mediated SDF-1/ CXCR4 regulatory axis. 16In mouse models with type 1 diabetes, an SMF balanced the activity of bone cells with the management of iron metabolism and redox state, resulting in a higher bone quality. 17SMFs have also illustrated increased osteogenic differentiation via Smad4 higher expression. 18Electrodynamic interactions, magneto-mechanical interactions and influences on electronic spin states are possible physical processes of SMF and its physiological consequences. 1Moreover, in an in-vivo study, the histological examination showed increased blood vessel generation within bone development after a SPION-loaded gelatin sponge administration. 19[22] The present study, for the first time, aimed to develop a microtissue involving Human umbilical vein endothelial cells (HUVECs) (representing the endothelial cells) and MG-63s (representing the osteoblasts) cell lines in combination with Fe 3 O 4 nanoparticles.This was to experiment with the osteogenesis and angiogenesis characteristics within this engineered bone microtissue.This kind of model would apply to comprehensive investigations and drug screening.

| Synthesis and preparation of iron oxide nanoparticles
The synthesis of nanoparticles (NPs) was carried out using the iron sources FeCl 2 •4H 2 O and FeCl 3 •6H 2 O (Merck), hydrochloric acid (HCl, 37%, Merck), deionized water (DI), and ammonia solution (NH 4 OH, 25% Merck) as the alkaline agent.The coprecipitation method was used to develop the SPIONs according to a procedure reported by Khodaei et al. 23 Two distinct 2 M ferrous and 1 M ferric solutions were initially prepared in diluted HCl (2 M).Then, 1 mL of the ferrous solution is added to 4 mL of the ferric solution and stirred for homogenization.Afterward, 50 mL of 0.4 M ammonia solution was added drop-wise with a rate of 0.75 mL/min under magnetic stirring.
Finally, the obtained black precipitate is decanted and washed with DI water several times.Before cell biology assessments, the SPIONs were autoclaved and remained sterile in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12, Gibco, USA) supplied by Penicillin/Streptomycin 1% (Bioidea, Iran).

| Characterization of nanoparticles
The crystalline structure of the nanoparticles was identified using x-ray diffraction (XRD-PW1800, Philips).The morphology of the nanoparticles was observed using a field emission scanning electron microscope (FE-SEM; TESCAN, Brno, Czech Republic), and the average nanoparticle size was determined using a transmission electron microscope (TEM; FEI Tecnai 12; Philips).A vibrating-sample magnetometer (VSM, Meghnatis Daghigh Kavir) was used to evaluate the magnetic properties of the synthesized nanoparticles.
A suspension containing 10,000 cells in DMEM media was added to each well from a 96-well Flat bottom plate.Before cell seeding, the wells were coated with a melted autoclaved solution of 1% agarosemedium (w/v) (100 μL/well) and cooled for 20 min.The cultured microtissues were examined under an optical microscope on the first, third and seventh days. 24,25

| Viability and mineralization assessment of the microtissue models
The viability of cells within all co-cultures microtissue groups was evaluated using the LIVE/DEAD assay, a fluorescent dye-based method.To stain both live and dead cells, 5 mg/mL of fluorescein diacetate (FDA) and 2 mg/mL of propidium iodide (PI, both Sigma) were re-suspended in a culture media without FBS.The microtissues were then incubated at room temperature for 5 min without light.
The samples were then examined using a fluorescence microscope (Olympus BX51).
The Alizarin Red staining method and analysis of ALP expression levels were employed to assess mineralization.The Alizarin Red staining process fixed all the groups in 70% ethanol for 20 min, followed by rinsing with PBS to remove any excess fixative.Afterward, the fixed groups were subjected to an Alizarin Red stain for 30 min.Alizarin Red is a dye that selectively binds to calcium ions, allowing for the visualization of mineralized deposits.
To quantify the extent of mineralization, the alizarin red stained cultures were incubated with 100 mM cetylpyridinium chloride, and optical density (OD) was measured using a spectrophotometer at a wavelength of 405 nm.
Alkaline phosphatase (ALP) activity was assessed using an ALP assay kit (Biorexfars, UK) following the instructions provided by the manufacturer.Diethanolamine Magnesium Chloride Preservative and P-Nitrophenyl phosphate were mixed at 75%:25% and 1000 μL was added to 20 μL of the samples.Afterward, the ALP levels were quantified by spectrophotometer at 405 nm.

| Determining MG-63 cells proliferation and mineralization upon SPION treatment
Four categories of 2D MG-63 cells treated with SPIONs were developed.SPION-free MG-63 cells were determined as the control group, and treated groups were exposed to 100, 50 and 25 μg/mL concentrations of SPIONs.Selecting the lower dosage range was based on previous reports illustrating cell toxicity of the higher doses of SPIONs. 26The experiments were applied on culture days three, five and seven.
To investigate the cell proliferation and toxicity of SPIONs in the different treatment groups, the 3-(4,5-dimethyl-2-thiazolyl)-2,5-dip henyl tetrazolium bromide (MTT) test (M5655, Sigma-Aldrich) was employed.Each well received 200 μL of a 0.5 mg/mL MTT solution, which was then incubated at 37°C for 4 h.Subsequently, 100 μL of dimethyl sulfoxide (Merck) replaced the first solution.Afterward, the ODs were measured with a plate reader spectrometer (FLUOstar Omega®, BMG Labtech) at 570 nm wavelength.To evaluate the mineralization of MG-63 cells after SPION administration, Alizarin Red S stain and APL levels were examined on days three, five and seven following the steps above.

| Scratch wound and transwell assays
After determining the optimum ratio of the HUVECs/MG-63s at 4:1, the scratch wound and transwell assays were performed to examine further the optimum SPION dosage treatment influencing cell migration and healing.A monolayer of 7 × 10 4 cells containing HUVECs and MG-63s (4:1) cultured in a 24-well plate was scratched by a 200 μL pipette.The detached cells were washed out using PBS and observed using optical microscopy.Residual cells were then cultured in SPOIN-free medium and different mediums containing SPOIN at 100, 50 and 25 μg/mL.After 24 h of incubation, the cells were evaluated using optical microscopy.

| Tube formation assay
This experiment evaluated the angiogenesis influenced by coculturing HUVECs with MG-63s and 50 μg/mL of SPION (based on the optimum dosage from the abovementioned observations).In this experiment, the co-cultured HUVECs and MG-63s at the ratio 4:1 treated with and without SPION (50 μg/mL) were compared to HUVECs alone. 1 × 10 5 cells were cultured in a 24-well plate coated with 2% neutralized COL-I (Sivan Cells Company, Iran).The PKH26 Red Fluorescent Cell Linker Kit (Sigma, USA) was used as general cell membrane labeling of HUVECs according to the manufacturer's instructions to track the angiogenesis.Tube formations were evaluated using an inverted microscope after they were incubated at 37°C for 6 h.Additionally, cells were fixed in 4% paraformaldehyde (Sigma-Aldrich, USA), and the nuclei of cells were stained with the blue fluorescent dye DAPI (4′,6-diamidino-2-phenylindole).The tube formation was examined by fluorescence microscope (Olympus BX51).

| Immunohistochemistry assessment
Immunohistochemistry (IHC) test was applied to observe CD31 expression as a marker of angiogenesis rate.HUVECs and MG-63s co-cultures at the ratio 4:1 treated with and without SPION (50 μg/mL), compared with HUVECs alone in the IHC test.In this regard, spheroids were washed using PBS after being fixed in 4% paraformaldehyde.Anti-CD31 antibody (dilution 1:50; Abcam) was mixed in all groups and incubated overnight at 4°C.The samples were subjected to a secondary antibody treatment the next day (dilution 1:200; Abcam).Then, the DAB + chromogen substrate system (dilution 1:50; Dako) and HRP (dilution 1:10,000; Abcam) were employed for its identification.A light microscope was used to identify it. 27

| Data analysis
To collect all the data, a minimum of three repeats were employed.

| Development and viability of the microtissues models
The bone microtissue models were established and characterized as previously described.Optical microscopy was employed to assess microtissue formation on Days 1, 3 and 7 of the culture (Figure 2).
Based on the study using Image J, the spheroid's average diameter was less than 400 μm, which is the ideal size for a spheroid to benefit from not having a discernible necrotic core. 28On the third day of culture, the cells underwent self-aggregation and the formation of compact microtissues was explicitly observed in the 4:1 HUVECs/ MG-63s ratio among the co-cultured groups.To qualitatively verify the microtissues' viability in different proportions of HUVECs and MG-63s, a LIVE/DEAD assay was applied.The MG-63 spheroids did not demonstrate high viability, so it was hypothesized that microtissue with endothelial cells might show higher viability levels.The results confirmed the highest FDA expression in the models with a 4:1 ratio of HUVECs to MG-63s (Figure 3).

| Quantification of osteogenesis using various ratios of cells in the microtissue model
Alizarin Red reaction and ALP expression level tests were conducted to assess the osteogenesis levels quantitatively.The results of the Alizarin Red reaction demonstrated a higher mineralization/calcification when the ratio of HUVECs to MG-63s was 4:1, and the difference was significant compared to HUVEC/MG-63 (3:2) (pvalue = 0.0436) (Figure 4).These findings were further supported by the results of ALP expression levels, which showed a noticeable increase in the HUVEC/MG-63 4:1 ratio group compared to other groups at ratios of HUVECs/MG-63s (3:2) (p-value = 0.0036) and HUVECs/MG-63s (4:1) (p-value = 0.0046) (Figure 4).

| Optimal concentration of SPION administration enhanced cell proliferation and osteogenesis
To investigate the effects of different concentrations of SPION on MG-63s, microtissue cell proliferation (MTT assay) and their mineralization (Alizarin Red reaction and ALP expression assays) were evaluated upon SPION administration.SPION at concentrations of 100, 50 and 25 μg/mL were compared to a free-SPION control group after 3, 5 and 7 days of treatment administration.Based on the MTT results, a concentration of 50 μg/mL of SPION exhibited a significant difference in cell proliferation compared to the control group on Day 5 (p-value < 0.0001) and Day 7 (p-value = 0.0069).In contrast, the 25 μg/mL concentration showed no noticeable difference compared to the control group on Days 3 and 7, while there was a significant difference on Day 5 (p-value = 0.0363).However, the concentration of 100 μg/mL of SPION led to a decline in cell proliferation levels, which indicates its toxicity to the cells (Figure 5).Administration of 50 μg/mL of SPION to MG-63s demonstrated the highest levels of calcium deposition on the 7th day, indicating its potency in promoting mineralization.This observation was confirmed by the microscopic examination of the cells and their ODs at 405 nm in the Alizarin Red experiment (p-value = 0.0069) (Figure 5).Additionally, the ALP expression test revealed that the osteoblasts exposed to 50 μg/mL of SPION exhibited significantly higher ALP expression levels compared to the control group on Day 3 (p-value = 0.0064), Day 5 (p-value = 0.0039) and Day 7 (pvalue = 0.0038) (Figure 5).

| SPION enhanced cells migrations and tubular formation
The evaluation of cell migration using scratch and tube formation assays was conducted.Fifteen microgram per milliliter of SPION incubated with the scratched co-culture of HUVECs-MG63s at 4:1 significantly increased the migration rate compared to other doses administration.
Furthermore, HUVECs co-cultured with MG-63s and 50 μg/mL of SPION could generate the highest cord-like structures on a collagen feeder, all conducting to an effective tube formation (Figure 7).

| SPION promoted angiogenesis
To investigate the angiogenesis, CD-31 expression, as a marker demonstrating the presence of endothelial cells, was checked in both SPION-treated and non-treated groups, including group 1: and HUVEC+ MG-63s (p = 0.0027) (Figure 7).In line with RT-PCR results, SPION exposure in the IHC test resulted in the appearance of angiogenesis in the SPION-treated groups (Figure 7).

| Osteogenesis-related gene expression levels
The levels of expression of osteogenesis markers, such as ALP (an early osteogenic marker of bone formation and bone calcification), COL-I (a collagenous protein in the bone matrix), and non-collagenous proteins like osteocalcin (OCN) and OPN in the bone matrix, were assessed.The treated groups were compared to the non-treated MG-63 control group as the representation of bone tissue's main element.It is demonstrated that SPION significantly boosts the OPN expression in the SPION-treated MG-63 group (p = 0.0006).Although no significant difference is shown between MG-63 plus HUVECs and MG-63 plus SPION groups (p = 0.0701), the OPN generation level is highest in the MG-63/HUVECs plus SPION group (p < 0.0001).In addition, OCN is illustrated to have a higher generation upon SPION application in the MG-63 group (p = 0.0006).Likewise, HUVECs co-culture showed a higher generation of this factor.There is no significant difference between the two later groups (p = 0.3315).Also, the MG-63/ HUVECs plus SPION group showed a particular increase in OCN expression compared to other groups (p < 0.0001).The ALP expression results show that although SPION and HUVEC both increase the expression of ALP in the MG-63 group, the presence of SPION has a more significant effect than that of HUVEC (p = 0.0037).Also, the company of SPION and HUVEC next to MG-63 compared to the MG group (p < 0.0001) and compared to the HUVEC+ MG group (p < 0.0001) increased ALP expression.The MG-63 plus SPION and MG-63 plus HUVECs groups had a higher expression of collagen type 1 than the control group, with no significant difference between the groups themselves (p = 0.552).Also, the synergistic effect of HUVEC+ MG-63+ SPION significantly increased COL-I expression compared to other groups.Overall, RT-PCR results showed a combination of HUVEC+ MG-63+ SPION leads to a synergic effect on the expression of osteogenesis markers (Figure 8).

| DISCUSS ION
While the bone tissue is generally capable of physiological regeneration and repair, managing substantial bone defects, particularly those resulting from tumours or infections, remains a significant challenge within the field of bone surgeries.These conditions may further lead to a severe delay in fusion or even non-fusion, endangering patients' muscle function.Typically, bone grafts from the patient's body (autologous) or a donor (allogeneic) have been used to repair significant bone defects.Nevertheless, these grafts can be both time-consuming and expensive to obtain.Therefore, a purposebuilt in-vitro bone model similar to the natural tissue may represent an optimal alternative to natural grafts. 29,30reover, 3D cell-culture systems are greatly anticipated to be dependable in vitro tools to bridge the gap between animal studies and human trials in various investigations.This development has reached a stage where the United States Food and Drug Administration (FDA) announced that it no longer necessitates animal testing as a prerequisite for approving human drug trial conduction. 31This study aimed to develop an effective preclinical bone tissue structure involving osteoblasts and endothelial to mimic osteogenesis and angiogenesis features of natural bone tissue.This kind of model would be applied to comprehensive investigations and drug screening.
Based on the live-dead, Alizarin Red reaction, ALP expression assays, tubular formation and IHC results in this investigation, 3D co-cultured HUVECs: MG-63s at a ratio of 4:1 results in the highest rate of viability, osteogenesis and angiogenesis.These data are supported by the fact that endothelial cells typically have a lower proliferation rate than osteoblasts.Due to this disparity, more HUVECs (endothelial cells) are needed to maintain a consistent interaction between osteoblast cells and endothelial cells within the co-culture system.This ensures that the angiogenic and osteogenic processes are balanced and synchronized, promoting optimal cell communication and tissue development. 32This is consistent with a previous study by Unger et al., which reported the co-culture of human dermal microvascular endothelial cells (HDMEC) and MG-63s (HDMEC: MG-63) in the ratios of 4:1 or 9:1 had sufficient quantities of both cell types after o1 week of cell culture.
In contrast, no endothelial cells were observed when the initial 1:1, 1:4 and 1:9 ratios were tested. 33Furthermore, as demonstrated in this study, the co-culture of HUVECs and MG63s cells at Additionally, MG-63 cells can secrete bone morphogenetic proteins (BMPs), which are potent inducers of osteoblast differentiation and bone formation. 36BMPs can also induce the expression of VEGF in endothelial cells, which can further promote angiogenesis and osteogenesis. 37Furthermore, in line with the results of this experiment, Dariima et al. verified that endothelial cells and osteoblasts may support tubule-like structures created by endothelial cells and that endothelial cells and osteoblasts synergistically boost the osteoblastic-related gene expression by osteoblasts.It is also suggested that the process improves the mean tubule length in coculture models. 38 further investigate how to enhance the viability, osteogenic potential and angiogenic properties of the bone tissue models, different concentrations of SPIONs were introduced into the samples.
The optimal effect of SPIONs was observed at 50 μg/mL.The toxicity of the SPIONs at a concentration of 100 μg/mL supports their dose-dependent nature, which is consistent with the findings of a study by Naqvi et al. that indicates the toxicity of the higher concentrations of SPIONs to murine macrophage cells. 39Furthermore, applying 50 μg/mL of SPION resulted in significant expression of osteogenesis markers in RT-PCR results.This was also indicated by calcium deposition observed through the Alizarin Red assay and ALP expression test.The increased activation of ALP following SPION administration is likely attributed to the de novo magnetic fields' properties, which trigger various cellular signalling pathways and facilitate their communication.This includes well-known pathways such as the mitogen-activated protein kinase [40][41][42] and the BMP signal pathways. 43,44This initially includes the over-generation of RUNX2, a hallmark of early osteogenic differentiation, which plays a significant role in several key signalling pathways that support osteogenesis. 45 the other hand, BMP2 overexpression can activate Smads proteins, leading to more RUNX2 expression.These shape the BMP2/Smads/RUNX2 signalling pathway, which is essential for bone formation. 46Notably, SPION can up-regulate INZEB2 levels, a crucial factor in maintaining osteogenesis because of its ability to down-regulate the ZEB2 levels.ZEB2 is a marker that can inhibit the BMP2/Smads/RUNX2 signalling pathway. 47These mechanisms elucidate the effect of MNPs on osteogenesis by markedly stimulating transforming growth factor β and WNT factors (Figure 9).9][50] Importantly, ALP plays a crucial role during the differentiation of osteoblasts.In addition, ALP is recognized as an early osteogenic marker involved in bone formation and calcification.Also, it is an enzyme that is secreted by osteoblasts, which are specialized bone-forming cells.
ALP plays a role in bone mineralization by providing a high phosphate concentration at the surface of the osteoblast cells.This phosphate is essential for depositing calcium and other minerals, facilitating the formation of hydroxyapatite, the main mineral Additionally, it enhanced bone regeneration by upregulating the osteogenic-related proteins Osterix and Runx-2. 54All these validate the unique physical, chemical and biological properties of SPIONs, making them an excellent option for biomedical applications in recent years.For instance, in a study on bone tissue regeneration, SPIONs have been shown to successfully increase the strength of the nanocomposite scaffold, promote calcium deposition and enhance ALP activity. 55,56eir impact on angiogenesis further confirmed the efficacy of SPIONs.The results from CD-31 expression in this study demonstrated a significant increase following the application of SPIONs.
These findings agree with an in-vivo survey where a gelatin sponge loaded with SPIONs showed increased bone mineral density and trabecular volume within the tissue.Notably, the histological examination revealed enhanced blood vessel formation concurrent with bone development.It was evident that osteoblasts and vascular endothelial cells had incorporated the SPIONs, resulting in heightened osteogenic and angiogenic capabilities. 19In another animal study by Singh et al. the development of new blood vessels alongside bone formation was observed when SPIONS were incorporated into polycaprolactone scaffolds. 57

| CON CLUS ION
Taken together, the direct contact between osteoblast and endothelial cells significantly affects the bone model viability and osteogenesis.The optimum ratio has been suggested to be 4:1 (HUVEC: MG-63).The samples' osteogenesis and angiogenesis further improved through 50 μg/mL SPION application, pointing out that it exerts its potential by up-regulating the osteogenesis factors involving OPN, OCN, ALP and COL-I as well as the angiogenesis factor CD-31 that is warranted to be confirmed by in vivo studies.
Ultimately, the findings of this study suggest how SPION, as an optimum component in the context of the engendered microtissue in this study, can enhance the models to replicate the physiological nature of bone in terms of osteogenic activity and angiogenic potential.This can improve the interaction with surrounding tissues and help promote orthopaedic implants with superior performance, preventing device failure.
Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to quantify the mRNA levels of collagen type I (COL1A1, NM_000088), osteocalcin (OCT, NM_199173), osteopontin (OPN, NM_000582), (ALP, NM_000478.6),β-actin (ACTB, NM_001101) and cluster of differentiation 31 (CD31, NM_000442.5).Reverse transcription and PCR amplification were used with the precise primers for each target gene.Real-time PCR equipment was used to measure the mRNA levels, and the cycle threshold values were used to conduct the analysis.An RNeasy Plus Mini Kit (QIAGEN) extracted the total RNA from the 3D models.The manufacturer's instructions used RevertAid H Minus First strand cDNA synthesis kit (Thermo Scientific, USA) to generate complementary DNA (cDNA).The cDNA allocations were kept at −20°C until further examination.Real-time reverse transcription polymerase chain reaction (RT-PCR) was carried out utilizing an Applied Biosystems StepOnePlusTM System (ABI, USA) and the SYBR® Premix Ex TaqTM II kit (Takara, Japan) to evaluate the relative gene expression.The reference gene B-ACTINE was utilized for standardization.Additionally, the 2-ΔΔCt technique was applied to calculate each gene's fold change in expression.

3 . 1 |
Error bars are used to illustrate standard deviation.Data were investigated by one-way analysis of variance (ANOVA) and Tukey's post hoc multiple comparison test through GraphPad Prism software (version 8.0.1;CA, USA).Statistical significance was defined as a pvalue of 0.05 or less.3| RE SULTS Characterization of nanoparticleThe x-ray diffraction pattern of the synthesized particles is illustrated in Figure 1A.Six major peaks can be distinguished at 20° of 30.06°, 35.57°, 43.0°, 53.77°, 57.05°, 62.77° and Coinciding with (111), (311), (002), (222), (400) and (220) crystalline planes, which are consistent with the diffraction pattern of Fe 3 O 4 according to the ICDD 96-900-5842 database.Morphological evaluation of the synthesized Fe 3 O 4 precipitate using FE-SEM (Figure 1B) and TEM (Figure 1C) images shows that the particles were almost spherical and had a relatively uniform size distribution.TEM images were further analysed with Image J software to determine the average nanoparticle diameter in the range of 5-7 nm.Finally, the magnetization curve of the obtained nanoparticle showed that the nanoparticles were entirely super-paramagnetic with a coercivity of close to zero Oe, and they have a saturation magnetization of 56.08 emu/g.

HUVECs and group 2 :
HUVECs+ MG 63s (4:1).Based on the RT-PCR results, the co-culture of HUVEC and MG increases the expression of the angiogenic gene CD-31 and has a significant difference with the SPION-free HUVECs (p < 0.0001), HUVECs+ SPION (p = 0.002)

F I G U R E 3 35 F I G U R E 5
Microtissues viability; Live (green)/dead (red) assay to determine live and dead cells proportion within four different ratios: MG-63s, HUVECs/MG-63s (3:2), HUVECs/MG-63s (2:1), and HUVECs/MG-63s (4:1) (scale bars: 100 μm).F I G U R E 4 Osteogenesis quantification: Alizarin red and alkaline phosphatase (ALP) tests in three HUVEC: MG-63 ratios showed the ratio of 4:1 has the highest levels of alizarin red and ALP secretion (*p < 0.05, **p < 0.01).the optimum ratio can facilitate cell interaction and the secretion of various factors that promote osteogenesis.This finding aligns with previous investigations that have shown a significant stimulation in endothelial growth factor (VEGF) and osteoblasts' differentiation and mineralization through the co-immobilization of Human osteoprogenitors with HUVECs. 34,Cell proliferation and osteogenesis levels upon SPION administration on the 3rd, 5th and 7th culture days; MTT results for cell proliferation levels (A).ALP activity levels (B).Osteogenic differentiation levels stained by alizarin red (scale bars: 500 μm) (C), Alizarin Red assay to quantitatively determine osteogenic levels (D) (*p < 0.05, **p < 0.01, ***p < 0.001).F I G U R E 6 Live (green)/dead (red) test in spheroids exposed to 50 μg/mL of SPION; HUVEC: MG-63 at a ratio of 4:1 has the highest viability compared to other groups (scale bars: 100 μm).